The escape of apoptosis is a major mechanism for cancer progression and drug resistance. The anti- apoptotic Bcl-2 family proteins, Bcl-2 and Bcl-xL, are among the most effective inhibitors of apoptosis and are frequently upregulated in various types of human cancer. The anti-apoptotic function of Bcl-2/Bcl-xL depends, at least in part, on their ability to heterodimerize with and inhibits the pro-apoptotic Bcl-2 family members Bax and Bak. Mutagenesis studies have shown that the conserved BH3 domain within the pro-apoptotic Bcl-2 family proteins is critical for their cell death-inducing function and also for their binding to Bcl-2 and Bcl-xL. Structural analyses of the Bcl-xL/BH3 complexes revealed that the a-helical BH3 peptides bind to the hydrophobic cleft formed by the BH1-3 domains of Bcl-xL. Moreover, it has been shown that synthesized peptides encompassing the BH3 domains of various pro-apoptotic Bcl-2 family members are sufficient to induce apoptosis of cancer cells. These studies suggest that targeting of Bcl-2/Bcl-xL by small molecule BH3- mimitics is a promising strategy for the development of a new class of anticancer drugs. To this end, we have rationally designed and synthesized a series of low-molecular-weight Bcl-xL antagonists based on the terphenyl and terephthalamide scaffolds to mimic the Bak-BH3 a-helix. Initial screens have identified several lead compounds, with binding affinities in the sub micromolar or high nanomolar region, by fluorescence polarization (FP) assay using fluoresceih-labeled Bak-BH3 peptide and purified Bcl-xL protein in vitro. Computational docking simulation and NMR analyses revealed that these synthetic inhibitors target the-BH3- binding cleft on the surface of Bcl-xL. Moreover, the active compounds can disrupt the interaction of Bcl-xL with Bax in vitro and in whole cells and cause cell death by apoptosis and tumor regression in nude mice. The goal of this project is to develop small molecules that induce apoptosis of cancer cells specifically by disrupting the BHS-mediated heterodimerization between pro- and anti-apoptotic proteins of the Bcl-2 family. The Specific Aims are: 1) To use rational design as well as high throughput screening (NTS) to identify BH3 a-helical mimics; 2) To determine the potency and specificity of the compounds identified under Specific Aim 1 to disrupt the BHS-mediated heterodimerization between pro- and anti-apoptotic members of the Bcl-2 family both in cell-free and in intact cells; 3) To determine the potency and selectivity of the Bcl/BH3 disrupters identified in Specific Aim 2 to induce apoptosis and to suppress tumor growth in cultured cells as well as in mouse models and verify the underlying biochemical mechanisms.